Programmed cell death is an important component of normal development, playing a major role in the formation of the nervous system, reproductive system, thymus and immune system. In addition, programmed cell death may contribute significantly to aging and to certain diseases such as AIDS where a subset of T lymphocytes is radically reduced in number. A major characteristic of programmed cell death is the compaction of chromatin accompanied by extensive fragmentation of the genome which may lead unalterably to cell. death. However, the mechanism of degradation is poorly understood. In addition, little is known about the genetic control of nuclear disintegration. We propose to examine these two issues in a unique model system where programmed nuclear death takes place reproducibly without cell death, where genetic mutants exist that affect nuclear disintegration, and where the process may be readily synchronized. In the protozoan Tetrahymena thermophila, which can be rapidly grown to high cell densities, macronuclear death occurs as a normal part of its easily inducible sexual reproduction. We will study the timing of DNA fragmentation in relationship to the two distinct stages of nuclear disintegration, examine the enzymatic means by which DNA is cleaved and how that might change with time. Probes for three different genomic entities, each with different characteristics, will be used to determine if disintegration is uniform or if greater complexity exists. We will examine these properties in mutant cells as compared to wild type cells while exploring the effects of various treatments on both. We will analyze the critical periods for gene expression related to the regulation of nuclear death, will investigate transcription in relationship to the stages of nuclear death and the differentiation of nuclei which replace the dying nucleus. Finally, we will identify genes which may play a role in the regulation of nuclear death using PCR amplified differential display of transcripts, selecting those PCR fragments which are unique to the critical periods of gene expression. These studies on this model system may contribute to our understanding of programmed cell death in higher organisms.